In cells of all organisms, DNA double-strand breaks are repaired by a process involving the action of RecA-class recombinases, helicases, nucleases, DNA polymerases, and DNA ligases. A classical pathway for double-strand break repair, called synthesis-dependent single strand annealing (SDSA), is shown in FIG. 1. Briefly, nucleases and helicases are used to unwind the DNA at the broken end and degrade the 5′-ending strand. The region of single-stranded DNA (with a terminal 3′ end) thus created is bound by a recombinase. The recombinase promotes a DNA strand invasion, to create a D-loop. The 3′ end of the invading DNA strand can be extended by DNA polymerase. If the invading strand is then separated from the invaded DNA, it can be joined to its cognate broken end via strand annealing. Replication and DNA ligation completes the repair process.
Although SDSA may be the most common pathway for double strand break repair, other variants either exist or have been proposed. All variants share the key steps of rendering a DNA single stranded by the action of helicases and nucleases, DNA strand invasion promoted by a RecA-family recombinase, extension of the invading DNA 3′ end with a DNA polymerase, and final ligation of nicks with DNA ligase.
What is needed in the art is a system for efficient repair of DNA double-strand breaks in vitro. Such a system will benefit multiple areas such as DNA genotyping in forensic science, DNA extraction from ancient sources, genome sequencing and metagenomics.